PROJECT SUMMARY/ABSTRACT Adoptive cell transfer (ACT) immunotherapies repurpose T cells from patients to specifically eliminate tumor cells by engineering them to express antigen-specific T cell receptors (TCRs) and chimeric antigen receptors (CARs). Currently, engineered T cell manufacturing is patient-specific and the timeline and cost of manufacture which may limit access to these new therapies. Some patients have a prognosis for survival that does not end beyond the inherent time required for cell manufacturing (on average 22 days for a current FDA-approved CAR- T product), and clinical responses are uneven and are related, at least partly, to the functional variability of the T cell composition of each product. This proposal seeks to remove these barriers by engineering human pluripotent stem cells (hPSCs) to generate non-alloreactive, antigen-specific mature, nave T cells using the Artificial Thymic Organoid (ATO) system, a novel in vitro method for efficiently and reproducibly producing mature, nave T cells from stem cell sources. CRISPR/Cas9 gene-editing will be used in hPSCs to generate insertion deletion mutations (INDELs) to knockout the beta-2-microglublin (B2M) gene, the critical subunit of all Class I MHC heterodimers, to prevent Class I MHC-mediated allo-rejection of T cells. To prevent the rearrangement of endogenous, and potentially allo-reactive TCRs, a multiplex CRISPR/Cas9 strategy will be used in hPSCs to completely excise the locus containing the recombination-activating-genes RAG1 and RAG2 in hPSCs. Together, these experiments will develop a platform for generating stem cell-derived, non-alloreactive mature nave T cells that could ultimately be applied toward an ?off-the-shelf? approach to T cell immunotherapy.